Methods for finding modulators of DYRK3 and DYRK2

ABSTRACT

Methods of finding agonists and antagonists to DYRK3 or DYRK2 are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to the earlier provisional U.S. application, Serial No. 60/326,973, which was filed on Oct. 4, 2001, the contents of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to methods of finding agonists and antagonists of human DYRK3, murine DYRK3, and human DYRK2.

BACKGROUND

[0003] A number of polypeptide growth factors and hormones mediate their cellular effects through a signal transduction pathway. Transduction of signals from the cell surface receptors for these ligands to intracellular effectors frequently involves phosphorylation or dephosphorylation of specific protein substrates by regulatory protein serine/threonine kinases (PSTK) and phosphatases. Serine/threonine phosphorylation is a major mediator of signal transduction in multicellular organisms. Receptor-bound, membrane-bound and intracellular PSTKs regulate cell proliferation, cell differentiation and signaling processes in many cell types. Aberrant protein serine/threonine kinase activity has been implicated or is suspected in a number of pathologies such as rheumatoid arthritis, psoriasis, septic shock, bone loss, many cancers and other proliferative diseases. Accordingly, serine/threonine kinases and the signal transduction pathways which they are part of are potential targets for drug design.

[0004] Recently, a mammalian kinase termed DYRK3 was shown to function as a suppressor of red cell development in both human and mouse bone marrow (Lord, et al., (2000) Blood 95, 2838-2846; Geiger, et al., (2001 Blood 97, 901-910). Antisense oligonucleotides directed against DYRK3 promote erythroid colony formation of bone marrow cells without affecting other colony forming units. In addition, DYRK3 is expressed predominantly in hematopoietic tissues, and within erythroid or erythropoietin (EPO)-responsive cells in particular. Together, this information suggests that inhibition of DYRK3 could serve as a treatment for anemia.

SUMMARY OF THE INVENTION

[0005] In one aspect, the instant invention relates to a method of identifying an agonist or antagonist of human DYRK2 (SEQ ID NO:6), said method comprising the steps of:

[0006] (a) incubating a candidate compound with human DYRK2 (SEQ ID NO:6); and

[0007] (b) determining the amount of phosphorylation of human EID-1 (SEQ ID NO:4) by human DYRK2 (SEQ ID NO:6); and

[0008] (c) identifying a compound that promotes the phosphorylation human EID-1 (SEQ ID NO:4) as an agonist of human DYRK2 (SEQ ID NO:6), and a compound that decreases the phosphorylation of human EID-1 (SEQ ID NO:4) as an antagonist of human DYRK2 (SEQ ID NO:6).

[0009] In a second aspect, the instant invention relates to a method of identifying an agonist or antagonist of DYRK3, said method comprising the steps of:

[0010] (a) incubating a candidate compound with a DYRK3 selected from group consisting of: murine DYRK2 (SEQ ID NO:3) and human DYRK3 (SEQ ID NO:2); and

[0011] (b) measuring the amount of phosphorylation of human EID-1 (SEQ ID NO:4) by said DYRK3; and

[0012] (c) identifying a compound that promotes the phosphorylation of human EID-1 (SEQ ID NO:4) as an agonist of said DYRK3 selected from group consisting of: murine DYRK2 (SEQ ID NO:3) and human DYRK3 (SEQ ID NO:2), and a compound that decreases the phosphorylation of human EID-1 (SEQ ID NO:4) as an antagonist of said DYRK3 selected from group consisting of: murine DYRK2 (SEQ ID NO:3) and human DYRK3 (SEQ ID NO:2).

[0013] In a third aspect, the instant invention relates to the above-recited method, wherein the DYRK3 is human DYRK3 comprising the amino acid sequence of residues 142-526 of SEQ ID NO:2.

[0014] In a fourth aspect, the instant invention relates to any of the above-recited methods, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using an antibody specific to phosphorylated human EID-1 (SEQ ID NO:4).

[0015] In a fifth aspect, the instant invention relates to any of the above-recited methods, wherein the human EID-1 is phosphorylated on serine 125 of human EID-1 (SEQ ID NO:4).

[0016] In a sixth aspect, the instant invention relates to any of the above-recited methods, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured by Liquid Chromatography-Electrospray Tandem Mass Spectrometry (LC-ES MS/MS).

[0017] In a seventh aspect, the instant invention relates to any of the above-recited methods, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using a labeled ATP.

[0018] In an eighth aspect, the instant invention relates to any of the above-recited methods, wherein the labeled ATP is P³³ labeled ATP.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows the nucleotide and amino acid sequences of human DYRK3 (SEQ NOs:1 and 2, respectively).

[0020]FIG. 2 shows the deduced amino acid sequence of murine DYRK3 (SEQ NO:3).

[0021]FIG. 3 shows the amino acid and nucleotide sequences of human EID-1 (SEQ NOs:4 and 5, respectively).

[0022]FIG. 4 shows the deduced amino acid sequence of human DYRK2 (SEQ ID NO:6).

DETAILED DESCRIPTION OF THE INVENTION

[0023] Given DYRK2 and DYRK3's potential involvement in erythropoiesis, it is of interest to discover activators and substrates therefor. As disclosed herein, human DYRK3 (hDYRK3) was used as the bait in a yeast two-hybrid screen (Fields, et al., (1989) Nature 340, 245-247) to identify human EID-1 as a protein which binds to and is an in vitro substrate of DYRK3. Human EID-1 was previously identified as an Rb and p300-binding protein, and inhibitor of differentiation [Miyake, et al., (200) Mol. Cell. Biol. 20, 8889-8902; MacLellan, et al., (2000) Mol. Cell. Biol. 20, 8903-8915]. Inhibitors of DYRK2 or DYRK3, in turn, decrease the phosphorylation of human EID-1 (SEQ ID NO:4). Compounds identified that inhibit the phosphorylation of human EID-1 may be useful as therapeutics for erythropoiesis.

[0024] The observation discussed below in Example 3 that either DYRK2 or DYRK3 can phosphorylate human EID-1 can be exploited to configure a screening method for identifying a compound which modulates the activity of DYRK3 or DYRK2. More particularly, the method comprises the step of incubating a candidate compound with DYRK3 or DYRK2, and determining the amount of phosphorylation on EID-1 by DYRK3 or DYRK2, whereby a compound which promotes the phosphorylation is an agonist of DYRK3 or DYRK2, and a compound which decreases the phosphorylation is an inhibitor of DYRK3 or DYRK2.

[0025] In a more preferred method, DYRK3 is a polypeptide of SEQ ID NO:2 or 3 or a polypeptide with amino acid sequence 142-526 of SEQ ID NO:2, and EID-1 is a polypeptide of SEQ ID NO:4; and DYRK2 is a polypeptide of SEQ ID NO:6. Yet in another preferred method, the amount of phosphorylation is measured using an antibody specific to phosphorylated EID-1 or using a labeled ATP, such as P³²- or P³³-ATP.

[0026] Using this data generated from the mass spectrometry, discussed below in Example 4, tools such as phosphospecific antibodies directed to a peptide derived from hEID-1 (SEQ ID NO:4) encompassing serine 125, or variants thereof, can be used to identify the phosphorylation state of this residue in cells or tissues expressing hDYRK3 (SEQ ID NO:2). In addition, one could evaluate the phosphorylation state of hEID-1 (SEQ ID NO:4) in cells or tissues treated with hDYRK3 agonists or inhibitors.

[0027] Definitions

[0028] The following definitions are provided to facilitate understanding of certain terms used frequently herein

[0029] “DYRK2” (or DYRK2 polypeptides) generally refers to human DYRK2 polypeptide having the amino acid sequence set forth in SEQ ID NO:6 or an allelic variant thereof.

[0030] “DYRK3” (or DYRK3 polypeptides) generally refers to human DYRK3 polypeptide having the amino acid sequence set forth in SEQ ID NO:2, or an allelic variant thereof, or to murine DYRK3 having the amino acid sequence set forth in SEQ ID NO:3.

[0031] “EID-1” (or EID-1 polypeptides) refers to human EID-1 polypeptide having the amino acid sequence set forth in SEQ ID NO:4 or an allelic variant thereof.

[0032] Polypeptides of the present invention also include variants of the aforementioned polypeptides. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acids are inserted, substituted, or deleted, in any combination.

[0033] Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NOs:2, 3, 4, or 6, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NOs:2, 3, 4, or 6. Preferred fragments are biologically active fragments that mediate the biological activity of DYRK2, DYRK3, or EID-1, including those with a similar activity or an improved activity, or with a decreased undesirable activity. For example, preferred fragments for DYRK3 include a polypeptide which has the amino acid sequence 142-526 of SEQ ID NO:2, which retains a catalytic activity.

[0034] Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation from naturally occurring sources, from genetically engineered host cells comprising expression systems or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.

[0035] “Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is “isolated” even if it is still present in said organism, which organism may be living or non-living.

[0036] “Polypeptide” refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well-described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, 1-12, in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter, et al., “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol, 182, 626-646, 1990, and Rattan, et al., “Protein Synthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci, 663, 48-62, 1992).

[0037] “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence, and preferably retains essentially the same biological function or activity as the reference polypeptide.

[0038] “Variant” refers to a polypeptide that differs from a reference polypeptide, and preferably retains the essential properties thereof. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, or deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of tyrosines, serines, and threonines and modification of C-terminal glycines.

[0039] “Identity” reflects a relationship between two or more polypeptide sequences, determined by comparing the sequences. In general, identity refers to an exact amino acid to amino acid correspondence of the two polypeptide sequences, respectively, over the length of the sequences being compared.

[0040] “% Identity”—For sequences where there is not an exact correspondence, a “% identity” may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting “gaps” in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

[0041] Methods for comparing the identity of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J, et al., Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wis., USA), BESTFIT and GAP, may be used to determine the % identity between two polypeptide sequences. BESTFIT uses the “local homology” algorithm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482-489, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to comparing two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison, GAP aligns two sequences, finding a “maximum similarity”, according to the algorithm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters “Gap Weight” and “Length Weight” used in each program are 12 and 4 for polypeptide sequences, respectively. Preferably, % identities are determined when the two sequences being compared are optimally aligned.

[0042] Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S. F., et al., J Mol Biol, 215, 403-410, 1990, Altschul, S. F., et al., Nucleic Acids Res., 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Md., USA and accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448,1988, available as part of the Wisconsin Sequence Analysis Package).

[0043] Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison.

[0044] Preferably, the program BESTFIT is used to determine the % identity of a query polypeptide sequence with respect to a reference polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.

[0045] “Identity Index” is a measure of sequence relatedness which may be used to compare a candidate sequence (polypeptide) and a reference sequence. Thus, for instance a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These differences may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polypeptide sequence having an Identity Index of 0.95 compared to a reference polypeptide sequence, an average of up to 5 in every 100 of the amino acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.

[0046] The relationship between the number of amino acid differences and the Identity Index may be expressed in the following equation:

n _(a) ≦x _(a)−(x _(a) ·I),

[0047] in which:

[0048] n_(a) is the number of amino acid differences,

[0049] x_(a) is the total number of amino acids in SEQ ID NO:2, 3, or 4,

[0050] I is the Identity Index,

[0051] · is the symbol for the multiplication operator, and

[0052] in which any non-integer product of x_(a) and I is rounded down to the nearest integer prior to subtracting it from x_(a).

BIOLOGICAL METHODS/EXAMPLES Example 1

[0053] Full-length human DYRK3 cDNA (SEQ ID NO: 1) was PCR amplified and cloned into pEG202 (Golemis, et al., in Current Protocols in Molecular Biology, John Wiley & Sons, New York (1994) using traditional molecular biology methods. The resulting plasmid, pEG202-DYRK3, encoded a fusion protein in which the DNA binding protein, LexA, was fused in-frame to DYRK3. The parent vector, pEG202, was a yeast expression vector which uses the alcohol dehydrogenase (ADH1) promoter to express the LexA fusion proteins and HIS3 as the selectable marker. All procedures, plasmids and strains used in the two-hybrid screen have been described in detail by Golemis, et al., supra. Yeast strain EGY48B (MATα, trp1, his3, ura3, 6ops-LEU2) was cotransformed with the plasmids pEG202-DYRK3 and pMW107. Transformants were selected using complete minimal media lacking uracil and histidine. The plasmid pPW107 is a yeast expression vector in which eight LexA operator sites are located upstream of a minimal GAL1 promoter which drives the expression of the LacZ gene, and URA3 as a selectable marker. Synthesis of the full length LexA-DYRK3 fusion was confirmed by Western blot analysis of yeast extracts using polyclonal antisera directed against LexA. It was confirmed that the LexA-DYRK3 fusion alone was unable to activate the LEU2 nor LacZ reporter strains.

[0054] Strain EGY48 ((MATα, trp1, his3, ura3, 6ops-LEU2) was transformed with a human fetal liver cDNA library in plasmid pJG4-5 using a library scale transformation protocol. This library plasmid contains the TRP1 selectable marker and allows the expression of cDNAs as fusions (AD fusions) to a cassette containing the SV40 nuclear localization sequence, the acid blob B42, and the hemagglutinin epitope tag. See Gyuris et al., Cell 75, 791-803 (1993). Expression of this fusion is under control of the galactose inducible promoter GAL1. Transformation reactions were plated onto complete minimal media lacking tryptophan. Approximately 4.5×10⁶ individual transformants were obtained, pooled and frozen. To ensure that each primary colony containing a library plasmid was mated, 2×10⁷ cells (approximately 3 times the number of individual transformants) were mixed with 2×10⁸ cells containing pEG202-DYRK3 and pMW107. The mixed cells were incubated on a nitrocellulose/YPD plate overnight at 30° C. to allow mating. Cells were washed off the nitrocellulose and plated onto minimal media lacking uracil, histidine, tryptophan and leucine with galactose/raffinose as the carbon source to induce expression of AD fusions. Colonies arising after 3 and 4 days of growth at 30° C. were picked to complete minimal media lacking uracil, histidine and tryptophan. Colonies containing potential interacting fusion proteins were then tested for galactose dependence and LacZ expression. Those isolates which activated both the LEU2 and LacZ reporters in a galactose dependent fashion were considered positive and pursued further.

[0055] Sequence analysis and subsequent GenBank searches using the BLASTX and BLATSN algorithms with the cDNA sequence or with the deduced amino acid sequence indicated that one of the AD fusion plasmids (isolate YFL11) encoded a protein identical retinoblastoma protein-associated protein (AF109873). Subsequent to this finding Miyake, et al. and MacLellan, et al. [MCB (2000 20, 8889-8902; MCB (2000) 20, 8903-8915] identified YFL11 as a protein which they term human EID-1 (SEQ ID NO:4).

Example 2

[0056] To determine whether DYRK3 and EID-1 interact in mammalian cells, murine DYRK3 (SEQ ID NO:3) was engineered for mammalian expression with an N-terminal cmyc6 tag in pCDNA3.1 (Invitrogen) and human EID-1 (SEQ ID NO:4) was engineered with an N-terminal V5-HIS6 tag in p423CDN, using standard molecular techniques. HEK293 cells were transfected with each construct together or singly using FUGENE6 transfection reagent (Roche Molecular Biochemicals). Two days after the transfection, the cells were washed twice in phosphate-buffered saline, and lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40) supplemented with 1×Complete protease inhibitor cocktail (Roche Molecular Biochemicals), 50 mM NaF, 10 mM β-glycerol phosphate, and 1 mM sodium vanadate. Lysates were clarified via centrifugation at 12,000×g for 5 min, and protein concentration determined using BCA protein reagent (Pierce). To immunoprecipitate, 1 ug of anti-V5 antibodies (Invitrogen) and 250 ul of IP buffer [50 mM Tris-HCl, pH7.5, 150 mM NaCl, 10% glycerol, 0.1% Nonidet P-40) supplemented with 1×Complete protease inhibitor cocktail (Roche Molecular Biochemicals)] was added to 300 ug of protein lysate and the reaction mixture incubated at 4° C. overnight. Protein A-Sepharose beads (100 ul of 50% slurry, Life Technologies) were added and incubation continued for 45 min. The beads were washed three times in IP buffer and resuspended in 40 ul 2×SDS sample buffer+5 ul reducing agent (Novex). The material was then separated via SDS page, transferred to nitrocellulose and subjected to western blot analysis with anti-cmyc or anti-V5 antibodies using standard conditions. Western blot analysis indicated that V5-EID-1 was immunoprecipitated from HEK293 cells transfected with V5-EID-1 and that cmyc-mDYRK3 is immunoprecipitated only when V5-EID-1 was present. This data, together with the yeast two-hybrid data, indicate that DYRK3 and EID-1 proteins interact.

Example 3

[0057] To determine if human EID-1 can serve as a substrate for DYRK3 or DYRK2, HEK 293 cells were transfected with p423CDN-V5-EID-1, and V5-EID-1 was immunoprecipitated as described in Example 2. The immunoprecipitated material was then subjected to western blot analysis to confirm the immunoprecipitation and an in vitro kinase reaction with purified DYRK3 or DYRK2 protein. To purify DYRK3, the catalytic domain (amino acids 142-526 from SEQ ID NO:2) was engineered for expression from a Baculovirus expression system with an N-terminal GST-tag and C-terminal HIS6-tag. Following infection of SF9 cells with virus, protein lysate was first passed over a glutathione-sepharose column followed by elution with 10 mM glutathione. Partially purified DYRK3 was then passed over a nickel column and eluted with 250 mM imidazole. To purify DYRK2, the protein with amino acid of SEQ ID NO:6 was engineered for expression from a Baculovirus expression system with an N-terminal HIS6-tag. Following infection of SF9 cells with virus, protein lysate was passed over a nickel column and eluted with 250 mM imidazole. To perform the in vitro kinase assay, immunoprecipitated V5-EID-1 or vector alone was incubated with kinase buffer containing 100 ng DYRK3 or 50 ng DYRK2 in 25 mM HEPES, pH 7.5, 10 mM MgCl₂, 10 mM β-glycerophosphate, 0.2 mM sodium orthovanadate, 50 uM adenosine triphosphate (ATP) and 5 uCi γ-³³P-ATP at 37° C. for 15 minutes. The reaction was terminated by the addition of sample buffer and boiling followed by electrophoresis and exposure to a Phosphorimager Screen (Molecular Dynamics, Sunnyvale, Calif.). There is a radioactive band that migrates at the same position as the EID-1 protein in the western blot while this band is absent in the vector alone lane in both the kinase reaction and western blot. This data demonstrates that EID-1 is phosphorylated by DYRK3 and DYRK2 in vitro.

[0058] In conclusion, the observation that either DYRK2 or DYRK3 can phosphorylate human EID-1 can be exploited to configure a screening method for identifying a compound which modulates the activity of DYRK3 or DYRK2. More particularly, the method comprises the step of incubating a candidate compound with DYRK3 or DYRK2, and determining the amount of phosphorylation on EID-1 by DYRK3 or DYRK2, whereby a compound which promotes the phosphorylation is an agonist of DYRK3 or DYRK2, and a compound which decreases the phosphorylation is an inhibitor of DYRK3 or DYRK2.

[0059] In a more preferred method, DYRK3 is a polypeptide of SEQ ID NO:2 or 3 or a polypeptide with amino acid sequence 142-526 of SEQ ID NO:2, and EID-1 is a polypeptide of SEQ ID NO: 4; and DYRK2 is a polypeptide of SEQ ID NO:6. Yet in another preferred method, the amount of phosphorylation is measured using an antibody specific to phosphorylated EID-1 or using a labeled ATP, such as P³²- or P³³-ATP.

Example 4

[0060] Human EID-1 (SEQ ID NO:4) was expressed in E. coli as an N-terminal HIS6 fusion and the protein purified by traditional methods known to those skilled in the art. To identify the site(s) on hEID-1 that are phosphorylated by hDYRK3 (SEQ ID NO: 2), an in vitro kinase assay was performed as follows: 10 μg purified EID-1 was incubated with kinase buffer containing 600 ng recombinant hDYRK3 in 44 mM HEPES, pH 7.5, 20 mM MgCl₂, 7 mM β-glycerophosphate, 0.14 mM sodium orthovanadate, 70 μM EDTA, 0.0018% Tween-20, 9.6 mM β-mercaptoethanol, 50 μM adenosine triphosphate (ATP) with or without 5 μCi g-33P-ATP at room temperature for 1 hr in a 100 μl reaction. The reaction was terminated by the addition of 0.1% TFA followed by electrophoresis and exposure to a Phosphorimager Screen (Molecular Dynamics, Sunnyvale, Calif.). In the reaction utilizing radioactivity, there was a radioactive band that migrated at the same position as the EID-1 protein following electrophoresis. The corresponding region in the lane that did not contain radioactivity was cut out of the gel and digested with Asp-N followed by Liquid Chromatography-Electrospray Tandem Mass Spectrometry (LC-ES MS/MS) to detect the site of phosphorylation. LC-ES MS/MS is a method that is well understood by those of skill in the art of mass spectrometry. A single phosphorylation site was identified on serine 125 of hEID-1 in the peptide DYPEEEQLSGAGYRVSAALEEA (SEQ ID NO:7). Using this information, tools such as phosphospecific antibodies directed to a peptide derived from hEID-1 (SEQ ID NO:4) encompassing serine 125, or variants thereof, can be used to identify the phosphorylation state of this residue in cells or tissues expressing hDYRK3 (SEQ ID NO:2). In addition, one could evaluate the phosphorylation state of hEID-1 (SEQ ID NO:4) in cells or tissues treated with hDYRK3 agonists or inhibitors.

[0061] All publications including, but not limited to, patents and patent applications, cited in this specification, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

[0062] The above description fully discloses the invention, including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Therefore, the examples provided herein are to be construed as merely illustrative and are not a limitation of the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

1 7 1 2061 DNA Homo sapiens 1 ggagcgaaat gcgctgagct gcagtgtctg gtcgagagta cccgtgggag cgtcgcgccg 60 cggaggcagc cgtcccggcg taggtggcgt ggccgaccgg acccccaact ggcgcctctc 120 cccgcgcggg gtcccgagct aggagatggg aggcacagct cgtgggcctg ggcggaagga 180 tgcggggccg cctggggccg ggctcccgcc ccagcagcgg aggttggggg atggtgtcta 240 tgacaccttc atgatgatag atgaaaccaa atgtcccccc tgttcaaatg tactctgcaa 300 tccttctgaa ccacctccac ccagaagact aaatatgacc actgagcagt ttacaggaga 360 tcatactcag cactttttgg atggaggtga gatgaaggta gaacagctgt ttcaagaatt 420 tggcaacaga aaatccaata ctattcagtc agatggcatc agtgactctg aaaaatgctc 480 tcctactgtt tctcagggta aaagttcaga ttgcttgaat acagtaaaat ccaacagttc 540 atccaaggca cccaaagtgg tgcctctgac tccagaacaa gccctgaagc aatataaaca 600 ccacctcact gcctatgaga aactggaaat aattaattat ccagaaattt actttgtagg 660 tccaaatgcc aagaaaagac atggagttat tggtggtccc aataatggag ggtatgatga 720 tgcagatggg gcctatattc atgtacctcg agaccatcta gcttatcgat atgaggtgct 780 gaaaattatt ggcaagggga gttttgggca ggtggccagg gtctatgatc acaaacttcg 840 acagtacgtg gccctaaaaa tggtgcgcaa tgagaagcgc tttcatcgtc aagcagctga 900 ggagatccgg attttggagc atcttaagaa acaggataaa actggtagta tgaacgttat 960 ccacatgctg gaaagtttca cattccggaa ccatgtttgc atggcctttg aattgctgag 1020 catagacctt tatgagctga ttaaaaaaaa taagtttcag ggttttagcg tccagttggt 1080 acgcaagttt gcccagtcca tcttgcaatc tttggatgcc ctccacaaaa ataagattat 1140 tcactgcgat ctgaagccag aaaacattct cctgaaacac cacgggcgca gttcaaccaa 1200 ggtcattgac tttgggtcca gctgtttcga gtaccagaag ctctacacat atatccagtc 1260 tcggttctac agagctccag aaatcatctt aggaagccgc tacagcacac caattgacat 1320 atggagtttt ggctgcatcc ttgcagaact tttaacagga cagcctctct tccctggaga 1380 ggatgaagga gaccagttgg cctgcatgat ggagcttcta gggatgccac caccaaaact 1440 tctggagcaa tccaaacgtg ccaagtactt tattaattcc aagggcatac cccgctactg 1500 ctctgtgact acccaggcag atgggagggt tgtgcttgtg gggggtcgct cacgtagggg 1560 taaaaagcgg ggtcccccag gcagcaaaga ctgggggaca gcactgaaag ggtgtgatga 1620 ctacttgttt atagagttct tgaaaaggtg tcttcactgg gacccctctg cccgcttgac 1680 cccagctcaa gcattaagac acccttggat tagcaagtct gtccccagac ctctcaccac 1740 catagacaag gtgtcaggga aacgggtagt taatcctgca agtgctttcc agggattggg 1800 ttctaagctg cctccagttg ttggaatagc caataagctt aaagctaact taatgtcaga 1860 aaccaatggt agtatacccc tatgcagtgt attgccaaaa ctgattagct agtggacaga 1920 gatatgccca gagatgcata tgtgtatatt tttatgatct tacaaacctg caaatggaaa 1980 aaatgcaagc ccattggtgg atgtttttgt tagagtagac tttttttaaa caagacaaaa 2040 catttttata tgattataaa a 2061 2 587 PRT Homo sapiens 2 Met Gly Gly Thr Ala Arg Gly Pro Gly Arg Lys Asp Ala Gly Pro Pro 1 5 10 15 Gly Ala Gly Leu Pro Pro Gln Gln Arg Arg Leu Gly Asp Gly Val Asp 20 25 30 Thr Phe Met Met Ile Asp Glu Thr Lys Cys Pro Pro Cys Ser Asn Val 35 40 45 Leu Cys Asn Pro Ser Glu Pro Pro Pro Pro Arg Arg Leu Asn Met Thr 50 55 60 Thr Glu Gln Phe Thr Gly Asp His Thr Gln His Phe Leu Asp Gly Gly 65 70 75 80 Glu Met Lys Val Glu Gln Leu Phe Gln Glu Phe Gly Asn Arg Lys Ser 85 90 95 Asn Thr Ile Gln Ser Asp Gly Ile Ser Asp Ser Glu Lys Cys Ser Pro 100 105 110 Thr Val Ser Gln Gly Lys Ser Ser Asp Cys Leu Asn Thr Val Lys Ser 115 120 125 Asn Ser Ser Ser Lys Ala Pro Lys Val Val Pro Leu Thr Pro Glu Gln 130 135 140 Ala Leu Lys Gln Tyr Lys His His Leu Thr Ala Tyr Glu Lys Leu Glu 145 150 155 160 Ile Ile Asn Tyr Pro Glu Ile Tyr Phe Val Gly Pro Asn Ala Lys Lys 165 170 175 Arg His Gly Val Ile Gly Gly Pro Asn Asn Gly Gly Tyr Asp Asp Ala 180 185 190 Asp Gly Ala Tyr Ile His Val Pro Arg Asp His Leu Ala Tyr Arg Tyr 195 200 205 Glu Val Leu Lys Ile Ile Gly Lys Gly Ser Phe Gly Gln Val Ala Arg 210 215 220 Val Tyr Asp His Lys Leu Arg Gln Tyr Val Ala Leu Lys Met Val Arg 225 230 235 240 Asn Glu Lys Arg Phe His Arg Gln Ala Ala Glu Glu Ile Arg Ile Leu 245 250 255 Glu His Leu Lys Lys Gln Asp Lys Thr Gly Ser Met Asn Val Ile His 260 265 270 Met Leu Glu Ser Phe Thr Phe Arg Asn His Val Cys Met Ala Phe Glu 275 280 285 Leu Leu Ser Ile Asp Leu Tyr Glu Leu Ile Lys Lys Asn Lys Phe Gln 290 295 300 Gly Phe Ser Val Gln Leu Val Arg Lys Phe Ala Gln Ser Ile Leu Gln 305 310 315 320 Ser Leu Asp Ala Leu His Lys Asn Lys Ile Ile His Cys Asp Leu Lys 325 330 335 Pro Glu Asn Ile Leu Leu Lys His His Gly Arg Ser Ser Thr Lys Val 340 345 350 Ile Asp Phe Gly Ser Ser Cys Phe Glu Tyr Gln Lys Leu Tyr Thr Tyr 355 360 365 Ile Gln Ser Arg Phe Tyr Arg Ala Pro Glu Ile Ile Leu Gly Ser Arg 370 375 380 Tyr Ser Thr Pro Ile Asp Ile Trp Ser Phe Gly Cys Ile Leu Ala Glu 385 390 395 400 Leu Leu Thr Gly Gln Pro Leu Phe Pro Gly Glu Asp Glu Gly Asp Gln 405 410 415 Leu Ala Cys Met Met Glu Leu Leu Gly Met Pro Pro Pro Lys Leu Leu 420 425 430 Glu Gln Ser Lys Arg Ala Lys Tyr Phe Ile Asn Ser Lys Gly Ile Pro 435 440 445 Arg Tyr Cys Ser Val Thr Thr Gln Ala Asp Gly Arg Val Val Leu Val 450 455 460 Gly Gly Arg Ser Arg Arg Gly Lys Lys Arg Gly Pro Pro Gly Ser Lys 465 470 475 480 Asp Trp Gly Thr Ala Leu Lys Gly Cys Asp Asp Tyr Leu Phe Ile Glu 485 490 495 Phe Leu Lys Arg Cys Leu His Trp Asp Pro Ser Ala Arg Leu Thr Pro 500 505 510 Ala Gln Ala Leu Arg His Pro Trp Ile Ser Lys Ser Val Pro Arg Pro 515 520 525 Leu Thr Thr Ile Asp Lys Val Ser Gly Lys Arg Val Val Asn Pro Ala 530 535 540 Ser Ala Phe Gln Gly Leu Gly Ser Lys Leu Pro Pro Val Val Gly Ile 545 550 555 560 Ala Asn Lys Leu Lys Ala Asn Leu Met Ser Glu Thr Asn Gly Ser Ile 565 570 575 Pro Leu Cys Ser Val Leu Pro Lys Leu Ile Ser 580 585 3 551 PRT Mus musculus 3 Met Met Ile Asp Glu Thr Lys Gly Pro Pro Tyr Ser Asp Thr Phe Ser 1 5 10 15 Asn Pro Ser Glu Ala Pro Val Ser Arg Arg Leu Asn Ile Thr Thr Glu 20 25 30 Pro Leu Thr Arg Gly His Thr Gln His Phe Val Asn Gly Ser Glu Met 35 40 45 Lys Val Glu Gln Leu Phe Gln Glu Phe Gly Asn Arg Arg Ser Asn Thr 50 55 60 Leu Gln Ser Asp Gly Ile Ser Asn Ser Glu Lys Ser Ser Pro Ala Ser 65 70 75 80 Gln Gly Lys Ser Ser Glu Ser Leu Ser Ala Val Lys Cys Asn Leu Ser 85 90 95 Ser Arg Pro Ser Lys Val Leu Pro Leu Thr Pro Glu Gln Ala Leu Lys 100 105 110 Gln Tyr Lys His His Leu Thr Ala Tyr Glu Lys Leu Glu Ile Val Ser 115 120 125 Tyr Pro Glu Ile Tyr Phe Val Gly Pro Asn Ala Lys Lys Arg Gln Gly 130 135 140 Val Ile Gly Gly Pro Asn Asn Gly Gly Tyr Asp Asp Ala Asp Gly Ala 145 150 155 160 Tyr Ile His Val Pro Arg Asp His Leu Ala Tyr Arg Tyr Glu Val Leu 165 170 175 Lys Ile Ile Gly Lys Gly Ser Phe Gly Gln Val Ala Arg Val Tyr Asp 180 185 190 His Lys Leu Arg Gln Tyr Val Ala Leu Lys Met Val Arg Asn Glu Lys 195 200 205 Arg Phe His Arg Gln Ala Ala Glu Glu Ile Arg Ile Leu Glu His Leu 210 215 220 Lys Lys Gln Asp Lys Thr Gly Ser Met Asn Val Ile His Met Leu Glu 225 230 235 240 Ser Phe Thr Phe Arg Asn His Val Cys Met Ala Phe Glu Leu Leu Ser 245 250 255 Ile Asp Leu Tyr Glu Leu Ile Lys Lys Asn Lys Phe Gln Gly Phe Ser 260 265 270 Val Gln Leu Val Arg Lys Phe Ala Gln Ser Ile Leu Gln Ser Leu Asp 275 280 285 Ala Leu His Lys Asn Lys Ile Ile His Cys Asp Leu Lys Pro Glu Asn 290 295 300 Ile Leu Leu Lys His His Gly Arg Ser Ala Thr Lys Val Ile Asp Phe 305 310 315 320 Gly Ser Ser Cys Phe Glu Tyr Gln Lys Leu Tyr Thr Tyr Ile Gln Ser 325 330 335 Arg Phe Tyr Arg Ala Pro Glu Ile Ile Leu Gly Cys Arg Tyr Ser Thr 340 345 350 Pro Ile Asp Ile Trp Ser Phe Gly Cys Ile Leu Ala Glu Leu Leu Thr 355 360 365 Gly Gln Pro Leu Phe Pro Gly Glu Asp Glu Gly Asp Gln Leu Ala Cys 370 375 380 Met Ile Glu Leu Leu Gly Met Pro Pro Gln Lys Leu Leu Glu Gln Ser 385 390 395 400 Lys Arg Ala Lys Tyr Phe Ile Asn Ser Lys Gly Leu Pro Arg Tyr Cys 405 410 415 Ser Val Ser Thr Gln Thr Asp Gly Arg Val Val Leu Leu Gly Gly Arg 420 425 430 Ser Arg Arg Gly Lys Lys Arg Gly Pro Pro Gly Ser Lys Asp Trp Ala 435 440 445 Thr Ala Leu Lys Gly Cys Gly Asp Tyr Leu Phe Ile Glu Phe Leu Lys 450 455 460 Arg Cys Leu Gln Trp Asp Pro Ser Ala Arg Leu Thr Pro Ala Gln Ala 465 470 475 480 Leu Arg His Pro Trp Ile Ser Lys Ser Thr Pro Lys Pro Leu Thr Met 485 490 495 Asp Lys Val Pro Gly Lys Arg Val Val Asn Pro Thr Asn Ala Phe Gln 500 505 510 Gly Leu Gly Ser Lys Leu Pro Pro Val Val Gly Ile Ala Ser Lys Leu 515 520 525 Lys Ala Asn Leu Met Ser Glu Thr Ser Gly Ser Ile Pro Leu Cys Ser 530 535 540 Val Leu Pro Lys Leu Ile Ser 545 550 4 187 PRT Homo sapiens 4 Met Ser Glu Met Ala Glu Leu Ser Glu Leu Tyr Glu Glu Ser Ser Asp 1 5 10 15 Leu Gln Met Asp Val Met Pro Gly Glu Gly Asp Leu Pro Gln Met Glu 20 25 30 Val Gly Ser Gly Ser Arg Glu Leu Ser Leu Arg Pro Ser Arg Ser Gly 35 40 45 Ala Gln Gln Leu Glu Glu Glu Gly Pro Met Glu Glu Glu Glu Ala Gln 50 55 60 Pro Met Ala Ala Pro Glu Gly Lys Arg Ser Leu Ala Asn Gly Pro Asn 65 70 75 80 Ala Gly Glu Gln Pro Gly Gln Val Ala Gly Ala Asp Phe Glu Ser Glu 85 90 95 Asp Glu Gly Glu Glu Phe Asp Asp Trp Glu Asp Asp Tyr Asp Tyr Pro 100 105 110 Glu Glu Glu Gln Leu Ser Gly Ala Gly Tyr Arg Val Ser Ala Ala Leu 115 120 125 Glu Glu Ala Asp Lys Met Phe Leu Arg Thr Arg Glu Pro Ala Leu Asp 130 135 140 Gly Gly Phe Gln Met His Tyr Glu Lys Thr Pro Phe Asp Gln Leu Ala 145 150 155 160 Phe Ile Glu Glu Leu Phe Ser Leu Met Val Val Asn Arg Leu Thr Glu 165 170 175 Glu Leu Gly Cys Asp Glu Ile Ile Asp Arg Glu 180 185 5 790 DNA Homo sapiens 5 gtcgacccac gcgtccgagc ggtttgcaca atgtcggaaa tggctgagtt gtccgagctg 60 tatgaagaga gcagtgacct gcagatggat gtgatgcctg gcgagggtga ccttccgcag 120 atggaggtag gcagcgggag ccgggagcta tccctgcgtc cctcccgcag cggggcccaa 180 cagctcgagg aggaaggccc aatggaggag gaggaggccc agccaatggc ggcgccagag 240 gggaaacgga gccttgctaa cgggcccaac gctggggagc agccaggcca ggtggcgggc 300 gcagacttcg agagcgagga cgagggcgag gaatttgatg actgggagga cgactacgac 360 tatcccgaag aggagcagct cagtggtgcc ggctacagag tatcagccgc tcttgaagaa 420 gccgacaaga tgtttctgag aacaagagaa ccagccctgg atggcgggtt tcagatgcat 480 tatgagaaga ccccgtttga tcagttagct tttatcgaag agcttttttc actgatggtt 540 gtcaatcgtc tgaccgaaga actcggctgt gatgagatta ttgatagaga gtagttagat 600 gctgttaaaa gaggaggaaa ctacttgagg agggacccaa ctttccgcta tcttttgggt 660 tcattccaaa tagttttgtg ccattgaaaa acttgacctt caaaaaaatt tgtttttcag 720 aatagaacac aataggacag tgactgcaca gttgtgaaaa aggaagagaa tcattaaaga 780 aaaaaaaaaa 790 6 528 PRT Homo sapiens 6 Met Asn Asp His Leu His Val Gly Ser His Ala His Gly Gln Ile Gln 1 5 10 15 Val Gln Gln Leu Phe Glu Asp Asn Ser Asn Lys Arg Thr Val Leu Thr 20 25 30 Thr Gln Pro Asn Gly Leu Thr Thr Val Gly Lys Thr Gly Leu Pro Val 35 40 45 Val Pro Glu Arg Gln Leu Asp Ser Ile His Arg Arg Gln Gly Ser Ser 50 55 60 Thr Ser Leu Lys Ser Met Glu Gly Met Gly Lys Val Lys Ala Thr Pro 65 70 75 80 Met Thr Pro Glu Gln Ala Met Lys Gln Tyr Met Gln Lys Leu Thr Ala 85 90 95 Phe Glu His His Glu Ile Phe Ser Tyr Pro Glu Ile Tyr Phe Leu Gly 100 105 110 Leu Asn Ala Lys Lys Arg Gln Gly Met Thr Gly Gly Pro Asn Asn Gly 115 120 125 Gly Tyr Asp Asp Asp Gln Gly Ser Tyr Val Gln Val Pro His Asp His 130 135 140 Val Ala Tyr Arg Tyr Glu Val Leu Lys Val Ile Gly Lys Gly Ser Phe 145 150 155 160 Gly Gln Val Val Lys Ala Tyr Asp His Lys Val His Gln His Val Ala 165 170 175 Leu Lys Met Val Arg Asn Glu Lys Arg Phe His Arg Gln Ala Ala Glu 180 185 190 Glu Ile Arg Ile Leu Glu His Leu Arg Lys Gln Asp Lys Asp Asn Thr 195 200 205 Met Asn Val Ile His Met Leu Glu Asn Phe Thr Phe Arg Asn His Ile 210 215 220 Cys Met Thr Phe Glu Leu Leu Ser Met Asn Leu Tyr Glu Leu Ile Lys 225 230 235 240 Lys Asn Lys Phe Gln Gly Phe Ser Leu Pro Leu Val Arg Lys Phe Ala 245 250 255 His Ser Ile Leu Gln Cys Leu Asp Ala Leu His Lys Asn Arg Ile Ile 260 265 270 His Cys Asp Leu Lys Pro Glu Asn Ile Leu Leu Lys Gln Gln Gly Arg 275 280 285 Ser Gly Ile Lys Val Ile Asp Phe Gly Ser Ser Cys Tyr Glu His Gln 290 295 300 Arg Val Tyr Thr Tyr Ile Gln Ser Arg Phe Tyr Arg Ala Pro Glu Val 305 310 315 320 Ile Leu Gly Ala Arg Tyr Gly Met Pro Ile Asp Met Trp Ser Leu Gly 325 330 335 Cys Ile Leu Ala Glu Leu Leu Thr Gly Tyr Pro Leu Leu Pro Gly Glu 340 345 350 Asp Glu Gly Asp Gln Leu Ala Cys Met Ile Glu Leu Leu Gly Met Pro 355 360 365 Ser Gln Lys Leu Leu Asp Ala Ser Lys Arg Ala Lys Asn Phe Val Ser 370 375 380 Ser Lys Gly Tyr Pro Arg Tyr Cys Thr Val Thr Thr Leu Ser Asp Gly 385 390 395 400 Ser Val Val Leu Asn Gly Gly Arg Ser Arg Arg Gly Lys Leu Arg Gly 405 410 415 Pro Pro Glu Ser Arg Glu Trp Gly Asn Ala Leu Lys Gly Cys Asp Asp 420 425 430 Pro Leu Phe Leu Asp Phe Leu Lys Gln Cys Leu Glu Trp Asp Pro Ala 435 440 445 Val Arg Met Thr Pro Gly Gln Ala Leu Arg His Pro Trp Leu Arg Arg 450 455 460 Arg Leu Pro Lys Pro Pro Thr Gly Glu Lys Thr Ser Val Lys Arg Ile 465 470 475 480 Thr Glu Ser Thr Gly Ala Ile Thr Ser Ile Ser Lys Leu Pro Pro Pro 485 490 495 Ser Ser Ser Ala Ser Lys Leu Arg Thr Asn Leu Ala Gln Met Thr Asp 500 505 510 Ala Asn Gly Asn Ile Gln Gln Arg Thr Val Leu Pro Lys Leu Val Ser 515 520 525 7 22 PRT Homo sapiens 7 Asp Tyr Pro Glu Glu Glu Gln Leu Ser Gly Ala Gly Tyr Arg Val Ser 1 5 10 15 Ala Ala Leu Glu Glu Ala 20 

We claim:
 1. A method of identifying an agonist or antagonist of human DYRK2 (SEQ ID NO:6), said method comprising the steps of: (a) incubating a candidate compound with human DYRK2 (SEQ ID NO:6); and (b) determining the amount of phosphorylation of human EID-1 (SEQ ID NO:4) by human DYRK2 (SEQ ID NO:6); and (c) identifying a compound that promotes the phosphorylation human EID-1 (SEQ ID NO:4) as an agonist of human DYRK2 (SEQ ID NO:6), and a compound that decreases the phosphorylation of human EID-1 (SEQ ID NO:4) as an antagonist of human DYRK2 (SEQ ID NO:6).
 2. A method of identifying an agonist or antagonist of DYRK3, said method comprising the steps of: (a) incubating a candidate compound with a DYRK3 selected from group consisting of: murine DYRK2 (SEQ ID NO:3) and human DYRK3 (SEQ ID NO:2); and (b) measuring the amount of phosphorylation of human EID-1 (SEQ ID NO:4) by said DYRK3; and (c) identifying a compound that promotes the phosphorylation of human EID-1 (SEQ ID NO:4) as an agonist of said DYRK3 selected from group consisting of: murine DYRK2 (SEQ ID NO:3) and human DYRK3 (SEQ ID NO:2), and a compound that decreases the phosphorylation of human EID-1 (SEQ ID NO:4) as an antagonist of said DYRK3 selected from group consisting of: murine DYRK2 (SEQ ID NO:3) and human DYRK3 (SEQ ID NO:2).
 3. The method as claimed in claim 2, wherein the DYRK3 is human DYRK3 comprising the amino acid sequence of residues 142-526 of SEQ ID NO:2.
 4. The method as claimed in claim 1, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using an antibody specific to phosphorylated human EID-1 (SEQ ID NO:4).
 5. The method as claimed in claim 4, wherein the human EID-1 is phosphorylated on serine 125 of human EID-1 (SEQ ID NO:4).
 6. The method as claimed in claim 4, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured by Liquid Chromatography-Electrospray Tandem Mass Spectrometry (LC-ES MS/MS).
 7. The method as claimed in claim 1, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using a labeled ATP.
 8. The method as claimed in claim 7, wherein the labeled ATP is P³³ labeled ATP.
 9. The method of claim 2, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using an antibody specific to phosphorylated human EID-1 (SEQ ID NO:4).
 10. The method as claimed in claim 9, wherein the human EID-1 is phosphorylated on serine 125 of human EID-1 (SEQ ID NO:4).
 11. The method as claimed in claim 9, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured by Liquid Chromatography-Electrospray Tandem Mass Spectrometry (LC-ES MS/MS).
 12. The method of claim 3, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using an antibody specific to phosphorylated human EID-1 (SEQ ID NO:4).
 13. The method as claimed in claim 12, wherein the human EID-1 is phosphorylated on serine 125 of human EID-1 (SEQ ID NO:4).
 14. The method as claimed in claim 12, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured by Liquid Chromatography-Electrospray Tandem Mass Spectrometry (LC-ES MS/MS).
 15. The method as claimed in claim 3, wherein the amount of phosphorylation of human EID-1 (SEQ ID NO:4) is measured using a labeled ATP.
 16. The method as claimed in claim 15, wherein the labeled ATP is P³³ labeled ATP. 